The research proposed is designed to identify specific genomic DNA target sequences which bind the mouse homeodomain protein Hox 1.4, a protein which may function as a transcription factor. The Hox 1.4 gene is expressed in the mid-gestation embryo and in meiotic and post-meiotic male germ cells. Our plan of attack includes a series of protein/DNA binding assays and a genetic selection protocol to identify genomic targets of Hox 1.4 protein binding. The first approach will utilize genomic DNA fragments ligated to polymerase chain reaction (PCR) """"""""catch linkers"""""""". The DNA will be incubated with the Hox 1.4 protein (produced in a Baculovirus expression system) to allow protein/DNA binding. DNA/protein complexes will be isolated by standard immunoselection protocols using anti-Hox 1.4 antibodies. The selected DNA is amplified by PCR, and protein binding and immunoselection are repeated to ensure isolation of specific sequences. The DNA of interest will be cloned, sequenced, and identified. In our second approach, Hox 1.4 DNA binding sequences (the homeodomain) are fused in frame with the transcription activator sequences of GAL4 and cloned into an expression plasmid with a selectable marker. Another plasmid has mouse genomic sequences cloned next to a reporter gene with a weak promoter. Co- transfection of the plasmids into yeast should result in clones producing the Hox 1.4/GAL4 fusion protein which will bind to the mouse genomic sequences and activate expression of the reporter gene. These sequences will be used as probes to identify the genes that Hox 1.4 binds. These experiments will identify specific DNA sequences which bind the Hox 1.4 protein and will enable us to determine which downstream genes may be regulated by Hox 1.4, providing information on molecular events and gene regulation during spermatogenesis.
